Doprinos poremećaja krvno-moždane barijere patofiziologiji Alchajmerove bolesti u transgenim animalnim modelima

Abstract

Krvno-moždana barijera (KMB) je ključna strukturna i funkcionalna prilagođenost krvnih sudova u centralnom nervnom sistemu, neophodna za njegovo normalno funkcionisanje. Osnovni zadatak KMB je da spreči slobodan prolazak humoralnih i hemijskih faktora i ćelija iz krvi u moždani parenhim i obrnuto. Periciti su ćelije koje naležu na endotelne ćelije krvnih kapilara, i zajedno sa endotelnim ćelijama, kao i proširenjima astrocitnih nastavaka i bazalnom membranom, čine KMB. Periciti, osim uloge u svim aspektima funkcionisanja i propustljivosti KMB, imaju ulogu u regulaciji protoka krvi, angiogeneze, čišćenja toksičnih materija iz mozga, neuroinflamaciji, kontrolišu ekspresiju proteina poreklom iz endotelnih ćelija koji ulaze u sastav adherentnih i čvrstih veza endotelnog sloja, a opisana su i njihova svojstva slična nervnim matičnim ćelijama. Alchajmerova bolest (AB) je neurodegenerativno oboljenje i najčešći oblik demencije kod ljudi starijih od 65 godina. AB se na neuropatološkom nalazu karakteriše akumulacijom vanćelijskog amiloida β i unutarćelijskog tau proteina u moždanom tkivu, kao i gubitkom neurona. Dodatno, istraživanja su pokazala da se promene u moždanoj cirkulaciji, protoku krvi i propustljivosti kapilara mogu primetiti i pre pojave kliničke slike sporadičnog oblika AB. Cilj ove teze je bio da se ispita integritet KMB i uloga percita u prisustvu i odsustvu AB patologije. Dodatni cilj je bio i da se ispita uloga PICALM proteina, jer se pokazalo da mutacije u ne-kodirajućem regionu PICALM gena mogu da predstavljaju faktor rizika za razvoj AB u kasnijem dobu (eng. late-onset). Uloga PICALM-a je praćena zasebno na endotelnim ćelijama i neuronima, a ispitana je i posledica farmakološkog povećanja ekspresije PICALM-a na nivo amiloida β. Konačno, ispitana je dijetalna restrikcija kao potencijalna intervencija u cilju smanjenja patologije vezane za AB. Da bi se ispitala uloga pericita korišćena su dva mišija modela: a) životinje koje imaju mutacije u receptoru PDGFRβ, koji je specifično eksprimiran na pericitima i vaskularnim glatkim mišićnim ćelijama i ima ključnu ulogu u regulaciji ćelijskog ciklusa, diferencijacije, rasta i razvoja i b) novouspostavljeni model akutnog gubitka pericita. Dodatno, da bi se ispitali poremećaji KMB u AB, korišćene su transgene životinje kao modeli AB - Tg2576, 3xTg i 5XFAD koje imaju jednu ili više mutacija u genima amiloidnog prekursorkog proteina i presenilina. Da bi se ispitale uloge PICALM-a u endotelnim ćelijama i neuronima, korišćeni su specijalni transgeni sojevi koji eksprimiraju Cre rekombinazu pod promotorom koji se specifično nalazi u endotelnim ćelijama (Cdh5-Cre) i neuronima (Camk2a-CreER), kako bi se osigurala specifična delecija PICALM-a iz ovih ćelija. Dijetalni režim ishrane svaki drugi dan (eng. every-other-day, EOD), je bio primenjen na ženke 5XFAD miševa. Svi eksperimenti su uključivali i odgovarajuće kontrolne životinje iz istog okota, a rezultati su prikupljeni analizom imunoblotova, imunohistohemijkih i histoloških bojenja, i testova ponašanja. Rezultati su pokazali da hronični gubitak pericita dovodi do demijelinizacije neuronskih nastavaka u beloj masi mozga, što dovodi do poremećaja u ponašanju, a da akutni gubitak pericita u adultnom životu može da dovede do gubitka nervnih ćelija u korteksu i hipokampusu i poremećaja u memoriji koja zavisi od normalne funkcije hipokampusa. Ispitivanje propustljivosti KMBumodelima AB je pokazalo, pored „klasične“ patologije – akumulacije amiloida, i izraženu narušenost KMB, čak i pre gubitka neurona. Dalje su rezultati otkrili da je PICALM protein izuzetno važan za čišćenje amiloida iz moždanog parenhima i da farmakološko povećanje ekspresije PICALM-a dovodi do smanjene količine amiloida u mozgu. Takođe, prisustvo PICALM-a u neuronima je važno za normalno ponašanje i zdravlje neurona. Primena dijetalne intervencije nije pokazala pozitivne efekte na smanjenje AB patologije. Naprotiv, ovakva intervencija je izazvala veću inflamaciju i dovela do drastičnog pada sinaptičkih proteina i čak smrti neurona kod ženki 5XFAD soja. Rezultati prikazani u ovoj disertaciji ukazuju na važnost održanja ne samo strukturnog integriteta KMB, već i optimalnog nivoa proteina koji ulaze u sastav ćelija KMB. Dodatno, rezultati ukazuju da promene na KMB mogu da dovedu do funkcionalnih promena i u odsustvu patologije izazvane Ab. Takođe, s obzirom da svi ispitani modeli AB imaju promene na KMB, važno je da se uključi i ova komponenta prilikom dijagnostifikovanja AB. Na kraju, iako je dijetalna restrikcija u prethodnim rezultatima pokazala uglavnom odlaganje ili umanjenje AB patologije; pol i starost jedinke, kao i tip i dužina trajanja dijetalne restrikcije treba pažljivo da se uzme u obzir pre odluke da se uvede kao tretman.

The blood-brain barrier (BBB) is a key structural and functional adaptation of blood vessels in the central nervous system, necessary for its normal functioning. The basic task of BBB is to prevent the free passage of humoral and chemical factors and cells from the blood into the brain parenchyma and vice versa. Pericytes are cells that attach to the endothelial cells of the blood capillaries, and together with the endothelial cells, as well as astrocytic end-feet and the basement membrane, constitute BBB. Pericytes, in addition to their role in all aspects of the functioning and permeability of BBB, play a role in regulation of blood flow, angiogenesis, clearance of toxic substances from the brain, neuroinflammation, control the expression of various proteins in endothelial cells, especially adherent and tight junctions proteins of the endothelial layer, and their properties similar to nerve stem cells are also described. Alzheimer's disease (AD) is a neurodegenerative disease and the most common form of dementia in people over 65 years of age. AD on neuropathological findings is characterized by accumulation of extracellular amyloid β and intracellular tau protein in brain tissue, as well as neuronal loss. In addition, studies have shown that changes in cerebral circulation, blood flow, and capillary permeability can be observed even before the onset of clinical presentation of sporadic AD. The aim of this thesis was to investigate the integrity of BBB and the role of pericytes in the presence and absence of AD pathology. Additional aim was to investigate the role of PICALM proteins, as it has been shown that mutations in the non-coding region of the PICALM gene may represent a risk factor for the development of late-onset AD. The role of PICALM was studied separately on endothelial cells and neurons, and the consequence of the pharmacological increase in PICALM expression to amyloid β levels was examined. Finally, dietary restriction was examined as a potential intervention to reduce ADrelated pathology. Two mouse models were used to examine the role of pericyte: a) animals that have mutations in the PDGFRβ receptor, which is specifically expressed on pericytes and vascular smooth muscle cells and plays a key role in cell cycle regulation, differentiation, growth and development, and b) newly established model of acute pericyte loss. Additionally, to examine KMB disorders in AD, transgenic animals were used as models of AD - Tg2576, 3xTg, and 5XFAD that have one or more mutations in the genes of amyloid precursor protein and presenilin. To examine the roles of PICALM in endothelial cells and neurons, special transgenic strains expressing Cre recombinase were used under a promoter specifically found in endothelial cells (Cdh5-Cre) and neurons (Camk2a-CreER), to provide specific deletion of PICALM from these cells. Every-other-day (EOD) diet regimen was administered to female 5XFAD mice. All experiments included appropriate control animals from the same litter, and the results were acquired by immunoblot analysis, immunohistochemical and histological staining, and behavioral tests. The results showed that chronic loss of pericytes leads to demyelination of neural processes in the brain white matter, which leads to behavioral deficits, and that acute loss of pericytes in adult brain can lead to loss of neurons in the cortex and hippocampus, and hippocampal-dependent memory deficits. Analysis of BBB permeability in AD models revealed, that in addition to "classical" pathology - amyloid accumulation, there is a significant BBB breakdown, which often occurs even before neuronal loss. The results further revealed that the PICALM protein is extremely important for the clearance of amyloid from the brain parenchyma and that the pharmacological increase in PICALM expression leads to a decreased level of amyloid in the brain. Also, the presence of PICALM in neurons is important for the normal behavior and neuronal health. And lastly, the use of dietary intervention showed no positive effects on the reduction of AB pathology. On the contrary, such an intervention caused greater inflammation and led to a drastic fall in synaptic proteins and even neuronal death in females of the 5XFAD strain. The results presented in this dissertation indicate the importance of maintaining not only the structural integrity of BBB, but also the optimal level of proteins expressed within different cells of BBB cells. In addition, the results indicate that changes in BBB can lead to functional changes even in the absence of pathology caused by Ab. Also, since all AD models tested in this study showed BBB breakdown, it is important to include this component when diagnosing AD. Lastly, although dietary restriction in the previous results showed a major delay or reduction of AD pathology; the sex and age of the individual as well as the type and length of dietary restriction should be carefully considered before deciding to introduce it as a treatment.